The long-term goal is to determine the coronary smooth muscle (CSM) adenosine A1 and P2Y2 receptor signaling mechanisms by which antagonism of the renin-angiotensin-aldosterone system (RAAS) and exercise training decrease coronary artery disease (CAD) and restenosis in metabolic syndrome (MetS). RAAS regulation of CSM A1 and P2Y2 receptor signaling that modulates kinases and downstream inflammation is unknown. Although drug-eluting stents decrease restenosis, further study is needed because of restenosis complications and progression of CAD adjacent to the stent, i.e. peri-stent CAD. Major findings in our previous grant provide outstanding rationale for this project. Ossabaw swine have clinically significant atherosclerosis (>50% stenosis) and we have for the first time stented natural lesions, not just balloon-injured healthy arteries. We cloned all 4 adenosine receptor isoforms (A1, A2A, A2B, A3) from pig and found A1 receptors (A1R) selectively expressed in CSM and increased in atherosclerotic and stented coronary. Direct activation of A1R induces CSM proliferation, indicating novel regulation. We cloned 3 porcine P2 nucleotide receptor isoforms and found the P2Y2 subtype is only expressed in diseased CSM and is selectively up-regulated in stented coronary. Overall hypothesis: in MetS RAAS (mainly aldosterone) increases coronary A1R and P2Y2R signaling, pivotal signals for CSM growth. The integrative experimental design compares lean vs. MetS Ossabaw RAAS antagonism. Atherosclerotic lesions are stented followed by recovery with or without exercise. Results are compared to simpler in vitro organ culture. Specific Aims are to test 6 specific hypotheses in MetS vs. lean Ossabaw: 1) Atherosclerosis, peri-stent CAD, and in-stent stenosis are increased in MetS and attenuated by RAAS antagonism and exercise. Intravascular ultrasound assesses CAD in vivo and optical imaging and histopathology assess CAD in vitro. 2) MetS increases molecular expression of the A1R and P2Y2R. Protein and mRNA will be determined in 5 different coronary segments: healthy, atherosclerotic, peri-stent, in-stent neointima, and in-stent media. 3) MetS increases functional expression of the A1R and P2Y2R by ERK, JNK, and AKT activation. Immunoblots and immunocytochemistry for phospho ERK, JNK, and AKT;store-operated Ca influx;and inflammation and oxidant stress quantify functional activation. 4) Angiotensin II and aldosterone directly, independently, and synergistically increase molecular and functional expression of A1R and P2Y2R, effects that are potentiated by dyslipidemia. In vivo treatments include several pig groups to be compared with in vitro organ culture. 5) Exercise will reverse the increased molecular and functional expression of A1R and P2Y2R in MetS. This Aim assesses more established CAD and stenting. 6) Blockade of A1R and P2Y2R attenuates in-stent stenosis in over-expansion injury and native atherosclerosis. Lean and MetS pigs will undergo in vivo coronary stenting with drug- and gene-eluting stents to selectively block A1R and P2Y2R. PUBLIC HEALTH RELEVANCE: Our studies involve highly clinically relevant coronary artery disease in the Ossabaw miniature pig, which superbly mimics complex metabolic syndrome ("pre-diabetes") and type 2 diabetes in humans. Thus, our findings will catalyze the translation of novel therapeutic interventions to humans, including bioengineered devices, drug-eluting stents (scaffold to open occluded artery), and systemic drug therapy. Drugs currently approved for humans are very likely to find new uses in treatment of coronary disease. We are uniquely positioned to benefit the prevention and regression of coronary disease, which will improve health in the U.S.